# Patent

## Title
BORIS isoforms and methods of detecting and treating disease

## Abstract

A method of detecting a proliferative disease, such as a disease associated with the abnormal expression of BORIS, in a mammal comprising testing for the expression of a BORIS isoform in the tissue of a mammal that does not express BORIS in the absence of disease, as well as a method of treating or preventing such a disease, isolated or purified BORIS isoform polypeptides and nucleic acids, and kits and arrays comprising same.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This patent application is a divisional of copending U.S. patent application Ser. No. 12/439,063, filed May 19, 2009, which claims the benefit of U.S. Provisional Patent Application No. 60/841,342, filed Aug. 31, 2006, which is incorporated by reference.
INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 161,782 bytes Byte ASCII (Text) file named “710345_ST25.txt,” created on May 2, 2012.


FIELD OF THE INVENTION
This invention pertains to BORIS isoform polypeptides and related compounds and compositions, and to the use of such polypeptides, compounds, and compositions for the detection and treatment of diseases associated with abnormal BORIS expression, such as cancer.
BACKGROUND OF THE INVENTION
The identification of tumor-associated antigens recognized by a mammalian immune system is useful for the diagnosis and treatment of cancer. A variety of tumor-associated antigens have been identified, including cancer/testis antigens that are expressed in cancer cells, but not in normal tissues other than testis. Only a minority of tumor-associated antigens, however, are immunogenic to the mammal that produces them.
BORIS (Brother of the Regulator of Imprinted Sites) is a tumor-associated antigen, which is activated in a wide range of human cancers. In fact, aberrant synthesis of the BORIS gene product has been found in over 300 primary tumors and cancer cell lines representing all major types of human cancers with recurrent 20q13 chromosomal gains. BORIS activation has also been found in all of the standard NCI-60 cancer cell lines, which are maintained by the National Cancer Institute (NCI), and which are thought to be a reasonably complete representative set of human cancers.
BORIS also is a CTCF paralog, which contains all eleven zinc fingers of CTCF, and has been shown to promote cell growth leading to transformation (see Loukinov et al, Proc. Natl. Acad. Sci (USA) 99, 6806-6811 (2002), and International Patent Application Publication WO 03/072799 (PCT/US03/05186)). BORIS has, therefore, also been referred to as “CTCF-like” or “CTCFL” protein. One mechanism of action by which BORIS is thought to cause cancer through interference with the maintenance of an appropriate methylation pattern in the genome mediated by CCCTC binding factor (CTCF) (see Klenova et al., Seminars in Cancer Biology 12, 399-414 (2002)). The BORIS gene is believed to map to the cancer-associated amplification region of chromosome 20q13.
The detection of aberrant expression of cancer markers, such as prostate specific antigen (PSA) and carcinoembryonic antigen (CEA), are known in the art. These assays, however, detect only a limited number of cancers and have limited positive predictive value for the detection or prognosis of new or recurring cancer. Accordingly, there is a need in the art to identify additional antigens whose expression can be linked to hyperproliferative diseases, such as cancer, as well as methods of detecting the presence of such antigens to aid in the detection, diagnosis, prognostication, or research of such disease states.
The invention provides methods and compositions useful for the detection, diagnosis, prognostication, or research of diseases associated with abnormal BORIS expression, such as cancer. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The invention provides a method of detecting a disease characterized by abnormal BORIS expression in a mammal, including but not limited to cancer, which method comprises testing for the expression of one or more BORIS isoforms in a tissue or body fluid of a mammal that does not express the BORIS isoform in the absence of disease.
The invention also provides a method of detecting a disease characterized by abnormal BORIS expression in a mammal, which method comprises testing for the expression of one or more BORIS isoform mRNA transcripts in a tissue or body fluid of a mammal that does not express the BORIS isoform in the absence of disease.
Also provided herein is a method of treating or preventing a disease associated with abnormal BORIS expression in a mammal. In one aspect, the method comprises administering a short interfering RNA (siRNA) molecule to a mammal afflicted with a disease associated with abnormal BORIS expression, wherein the siRNA molecule comprises a sequence of at least 10 contiguous nucleotides that is complimentary to a BORIS isoform mRNA transcript. In a related aspect, the method comprises administering an anti-BORIS antibody to a mammal afflicted with a disease associated with abnormal BORIS expression, wherein the anti-BORIS antibody selectively binds to a isoform polypeptide.
The invention additionally provides a method of inducing an immune response in a mammal comprising administering a BORIS isoform to the mammal.
The invention further provides an isolated or purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42, an isolated or purified nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-24, and a composition comprising such a polypeptide or nucleic acid.
In a related aspect, the invention provides a kit for the detection of BORIS expression in a mammal, which kit comprises (a) a probe set comprising one or more probes that bind to (i) a BORIS polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42, (ii) an auto-antibody to a BORIS polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42, or (iii) a BORIS isoform mRNA transcript comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-24, and (b) a reagent that facilitates the detection of the probe.
An array useful for the detection of BORIS expression in a mammal also is provided by the invention, the array comprising one or more probes immobilized on a substrate, wherein the probes bind to (i) a BORIS isoform comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42, (ii) an auto-antibody to a BORIS isoform comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42, or (iii) an BORIS isoform mRNA transcript comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-24.
The invention further provides a database comprising a BORIS expression profile of one or more different types of cancer, wherein the database facilitates the comparison of a BORIS expression profile of a patient with the BORIS expression profile of one or more different types of cancer.
Also provided by the invention is a method of inducing an immune response to BORIS in a mammal comprising administering to the mammal a BORIS isoform comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1D are illustrations depicting alternative splice variants expressed by the BORIS gene in the human testes.



DETAILED DESCRIPTION OF THE INVENTION
The BORIS polypeptide disclosed in Klenova et al., Seminars in Cancer Biology 12, 399-414 (2002) comprises the amino acid sequence provided herein as SEQ ID NO: 43, and is encoded by the mRNA sequence provided herein as SEQ ID NO: 44. This BORIS polypeptide, however, is only one of a family of polypeptides encoded by the BORIS gene. BORIS mRNA splice variants encoding BORIS and BORIS isoforms are described herein, including twenty-four specific examples of BORIS isoform mRNA transcripts that encode seventeen different BORIS isoform polypeptides. The exemplary BORIS isoform mRNA transcripts each comprise a nucleic acid sequence of SEQ ID NOs: 1-24, and the seventeen BORIS isoform polypeptides comprise a nucleotide sequence of SEQ ID NOs: 25-42. The BORIS isoform mRNA transcripts and the polypeptides encoded thereby are set forth in Table 1. In particular, the BORIS isoform mRNA transcripts comprising the nucleotide sequences of SEQ ID NOs: 1, 2, and 3 encode a polypeptide comprising an amino acid sequence identical to that of the previously disclosed BORIS polypeptide (e.g., SEQ ID NO: 43). Although these mRNA transcripts encode the same BORIS polypeptide, the mRNA transcripts are, themselves, alternative spice variants of the previously disclosed BORIS mRNA and, therefore, comprise different nucleotide sequences. The other BORIS isoform polypeptides comprise amino acid sequences that are different from the BORIS polypeptide previously disclosed in Klenova et al., supra.
For the purposes of describing the invention, the terms “BORIS” and “BORIS polypeptide” shall be used to refer to the BORIS polypeptide comprising the amino acid sequence of SEQ ID NO: 43, and the term “BORIS mRNA” shall be used to refer to an mRNA transcript having a nucleotide sequence of SEQ ID NO: 44.
The terms “BORIS isoform” and “BORIS isoform polypeptide” shall be used herein to refer to a polypeptide encoded by a splice variant mRNA transcript of the BORIS gene, which has an amino acid sequence that is different from SEQ ID NO: 43. Examples of BORIS isoform polypeptides include polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42. The term “BORIS isoform mRNA” shall be used to refer to an mRNA splice variant of the BORIS gene, which has a nucleotide sequence that is different from SEQ ID NO: 44. Examples of BORIS isoform mRNA transcripts include mRNA molecules that comprise a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42, or a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-24.
The term “BORIS gene” shall be used herein to refer to the genomic sequence of BORIS, which encodes the BORIS polypeptide as well as the BORIS mRNA splice variants (e.g., BORIS isoform mRNA transcripts) and BORIS isoform polypeptides.
As used herein, the term “isolated” means the removal of a nucleic acid or polypeptide molecule from its natural environment. The term “purified” means that a given nucleic acid or polypeptide molecule, whether it has been removed from nature or synthesized and/or amplified under laboratory conditions, has been increased in purity, wherein “purity” is a relative term, not “absolute purity.”
As used herein, the term “nucleic acid” is intended to encompass a polymer of DNA or RNA, (i.e., a polynucleotide), which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. Similarly, a “polypeptide” is intended to encompass a linear sequence of amino acids (i.e., a primary protein structure) of any length, as well secondary, tertiary, and quaternary protein structures, any of which can contain non-natural or altered amino acids.
Some aspects of the invention are described with reference to the use of an antibody. It is intended that the use of an antibody can be substituted by the use of an antibody fragment of any of the various known forms (e.g., F(ab)2′ fragments, single chain antibody variable region fragment (ScFv) chains, and the like). Thus, for the sake of brevity, term “antibody” as used herein is intended to encompass antibodies as well as antibody fragments. Wherever the term “antibody” is used, it is specifically contemplated that an antibody fragment can be used instead.
The term “selectively binds” as used herein means to bind to a target molecule (e.g., polypeptide or nucleic acid) with a greater affinity or with preference as compared to another polypeptide or nucleic acid. Thus, for instance, a probe selectively binds a target BORIS isoform mRNA transcript if it binds such target with preference or greater affinity than to a nucleic acid that is not a BORIS isoform mRNA transcript. Similarly, an anti-BORIS antibody selectively binds a BORIS isoform if it binds such target isoform with preference or greater affinity than to a polypeptide that is not a BORIS isoform. Selectively binds also can be used to mean that a molecule binds one BORIS isoform polypeptide or mRNA transcript with preference, or greater affinity, than another BORIS isoform polypeptide or mRNA transcript.
The invention provides a method of detecting a disease characterized by abnormal gene expression, such as a hyperproliferative disease involving abnormal BORIS expression (e.g., cancer) in a mammal. In this respect, “abnormal BORIS expression” means the expression of BORIS in the tissues or body fluids of a mammal that do not express the BORIS gene in the absence of disease, as discussed in greater detail herein.
According to one aspect of the invention, the method comprises testing for the expression of one or more BORIS isoforms in a tissue or body fluid of a mammal that does not express the BORIS isoform in the absence of disease. The one or more BORIS isoforms can, for example, comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42. In a related aspect of the invention, the method comprises testing for the expression of one or more BORIS isoform mRNA transcripts in a tissue or body fluid of a mammal that does not express the BORIS isoform mRNA in the absence of disease. The one or more BORIS isoform mRNA transcripts can comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-24. The expression of a BORIS isoform or BORIS isoform mRNA transcript, as described above, in such a tissue or fluid of the mammal is indicative of the presence of disease in the mammal.
The terms “testing” and “detecting” (and permutations thereof) as used herein mean to investigate or determine the presence of a condition. Thus, for instance, “testing for” or “detecting” the expression of a gene or gene product means to investigate or determine whether the gene is being expressed or the gene product is present.
Preferably, the method of detecting a disease comprises testing for the expression of more than one BORIS isoform. For instance, the method can comprise testing for the expression of two or more BORIS isoform polypeptides or BORIS isoform mRNA transcripts, preferably five or more, 10 or more, 15 or more, or even all of such BORIS isoform polypeptides or mRNA transcripts. Whether the method involves the detection of one BORIS isoform polypeptide or BORIS isoform mRNA transcript, or more than one of BORIS isoform polypeptides or BORIS isoform mRNA transcripts, the method of detecting a disease also can comprise testing for the expression of a BORIS polypeptide comprising the amino acid sequence of SEQ ID NO: 43, or an mRNA transcript comprising the nucleotide sequence of SEQ ID NO: 44.
The method of the invention can be used to detect any disease characterized by or associated with abnormal BORIS expression including, but not limited, to the detection of cancer. As mentioned above, BORIS mRNA has been detected in several hundred cancer and tumor cell lines representing most of the major forms of cancer. Thus, the method of the invention can be used to detect any type of cancer. Such cancers include, but are not limited to, cancer of the oral cavity and pharynx, the digestive system (e.g., the esophagus, stomach, small intestine, colon, rectum, anus, liver, gall bladder, and pancreas), the respiratory system (e.g., the larynx, lung, and bronchus, including non-small cell lung carcinoma), bones and joints (e.g., bony metastases), soft tissue, the skin (e.g., melanoma), breast, the genital system (e.g., the uterine cervix, uterine corpus, ovary vulva, vagina, prostate, testis, and penis), the urinary system (e.g., the urinary bladder, kidney, renal pelvis, and ureter), the eye and orbit, the brain and nervous system (e.g., glioma), or the endocrine system (e.g., thyroid). The cancer also can be a lymphoma (e.g., Hodgkin's disease and Non-Hodgkin's lymphoma), multiple myeloma, or leukemia (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and the like).
Furthermore, as demonstrated herein, not all cancers are associated with the expression of the same BORIS isoforms. Accordingly, by testing for the expression of one or more different BORIS isoforms, it is possible to generate a BORIS expression pattern that can be used to distinguish between different types of cancers, or to detect a specific type of cancer. Thus, the method of detecting a disease associated with abnormal BORIS expression preferably comprises a step of comparing the BORIS expression of the mammal to a control, which comparison can be used, for example, to classify the type of disease with which the mammal might be afflicted. Any suitable control can be used for this purpose. Typically, the control can be provided by a BORIS expression pattern corresponding to a particular type of disease or cancer (e.g., the BORIS expression pattern of a mammal known to be afflicted with a particular type of disease or cancer).
Testing for the expression of one or more BORIS isoforms or BORIS isoform mRNA transcripts can be performed using any suitable technique. Typically, a sample of the tissue or body fluid of the mammal that does not express the BORIS gene (i.e., does not express the BORIS polypeptide or any isoform thereof) in the absence of disease is obtained, and the sample is tested for the expression of a BORIS isoform or BORIS isoform mRNA transcript. The BORIS gene is normally expressed only in the testes and ovaries. Accordingly, a sample of any tissue or body fluid other than a testicular or ovarian tissue sample can be used. The sample can be a solid sample or the sample can be fluid, such as a sample of body fluid. For instance, a section of whole tissue can be used for immunohistochemistry-based analysis, or can be homogenized to liquefy the components found in the tissue. The sample preferably is a fluid. Suitable fluid samples include, but are not limited to, blood, serum, plasma, lymph, and interstitial fluid.
Testing for the expression of one or more BORIS isoforms can comprise, for example, directly detecting one or more BORIS isoform polypeptides, or detecting one or more mRNA transcripts that encode a BORIS isoform polypeptide. Suitable methods of detecting protein levels in a sample include Western Blotting, radio-immunoassay, and Enzyme-Linked Immunosorbant Assay (ELISA). Such methods are described in Nakamura et al., Handbook of Experimental Immunology, 4th ed., Wol. 1, Chapter 27, Blackwell Scientific Publ., Oxford, 1987. When detecting proteins in a sample using an immunoassay, the sample is typically contacted with antibodies or antibody fragments (e.g., F(ab)2′ fragments, single chain antibody variable region fragment (ScFv) chains, and the like) that specifically bind the target protein (e.g., BORIS isoform polypeptide). Thus, BORIS isoform polypeptides can be detected, for example, by contacting a sample of the tissue or body fluid of the mammal with an antibody or antibody fragment to the BORIS isoform, and detecting the binding of the antibody or antibody fragment with a BORIS isoform from the sample. Antibodies and other polypeptides suitable for detecting BORIS isoform polypeptides in conjunction with immunoassays are commercially available and/or can be prepared by routine methods, such as methods discussed elsewhere herein (e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Publishers, Cold Spring Harbor, N.Y., 1988).
The immune complexes formed upon incubating the sample with the antibody are subsequently detected by any suitable method. In general, the detection of immune complexes is well-known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. U.S. patents concerning the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.
For example, the antibody used to form the immune complexes can, itself, be linked to a detectable label, thereby allowing the presence of or the amount of the primary immune complexes to be determined. Alternatively, the first added component that becomes bound within the primary immune complexes can be detected by means of a second binding ligand that has binding affinity for the first antibody. In these cases, the second binding ligand is, itself, often an antibody, which can be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
Other methods include the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody, that has binding affinity for the first antibody can be used to form secondary immune complexes, as described above. After washing, the secondary immune complexes can be contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. A number of other assays are contemplated; however, the invention is not limited as to which method is used.
Similarly, mRNA transcripts encoding a BORIS isoform can be detected by any suitable technique. Typically, a sample of the tissue or body fluid of the mammal (e.g., the RNA material of such a sample) is contacted with a nucleic acid probe that binds to an mRNA transcript encoding a BORIS isoform, and the binding of the nucleic acid probe with a BORIS isoform from the sample is detected. Suitable methods of detecting or measuring mRNA include, for example, Northern Blotting, reverse-transcription PCR(RT-PCR), and real-time RT-PCR. Such methods are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1989. Of these methods, real-time RT-PCR is typically preferred. In real-time RT-PCR, which is described in Bustin, J. Mol. Endocrinology. 25: 169-193 (2000), PCRs are carried out in the presence of a labeled (e.g., fluorogenic) oligonucleotide probe that hybridizes to the amplicons. The probes can be double-labeled, for example, with a reporter fluorochrome and a quencher fluorochrome. When the probe anneals to the complementary sequence of the amplicon during PCR, the Taq polymerase, which possesses 5′ nuclease activity, cleaves the probe such that the quencher fluorochrome is displaced from the reporter fluorochrome, thereby allowing the latter to emit fluorescence. The resulting increase in emission, which is directly proportional to the level of amplicons, is monitored by a spectrophotometer. The cycle of amplification at which a particular level of fluorescence is detected by the spectrophotometer is called the threshold cycle, CT. This value is used to compare levels of amplicons. Probes suitable for detecting mRNA levels of the biomarkers are commercially available and/or can be prepared by routine methods, such as methods discussed elsewhere herein. Specific protocols for these and other methods of detecting polypeptides and mRNA transcripts in samples of mammalian tissues and body fluids are well known in the art (see, e.g., Sambrook et al., supra).
Alternatively, the expression of BORIS isoforms can be tested indirectly by detecting the presence of auto-antibodies to the BORIS isoforms in the mammal. Without wishing to be bound to any particular theory, it is believed that the abnormal expression of BORIS isoforms in tissues and body fluids where BORIS isoforms are not normally found (e.g., other than the testes or ovaries) causes the immune system of the mammal to produce antibodies to the BORIS isoforms that can be detected in a sample (e.g., the tissues, sera, or bloodstream) obtained from the diseased mammal. In the absence of such a disease, the BORIS isoforms are confined to the tissues and organs in which they are normally found in a non-diseased mammal, and the immune system of the mammal does not produce antibodies against the BORIS isoforms. Thus, a sample taken from a non-diseased mammal (e.g., a mammal without a disease characterized by abnormal BORIS expression) will not contain anti-BORIS isoform antibodies. Accordingly, by detecting the presence or absence of anti-BORIS isoform antibodies in the sample of a mammal, the method of the invention enables a determination as to whether the mammal has a disease characterized by abnormal BORIS expression, such as cancer.
Any suitable method of detecting anti-BORIS auto-antibodies in a sample can be used. Auto-antibodies to the BORIS isoforms can be detected, for instance, by contacting a sample of tissue or body fluid of the mammal with one or more BORIS isoforms or immunogenic portions thereof (e.g., one or more isolated or purified polypeptides comprising an immunogenic portion of the amino acid sequence of any of SEQ ID NOs: 25-42). The sample can be contacted with a BORIS polypeptide using any suitable method known in the art. Preferably, the sample is contacted with a BORIS polypeptide in vitro or ex vivo. In vitro and ex vivo methods for detecting antibodies in a sample are well known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA), affinity chromatography, and radioimmunoassay (RIA).
By “immunogenic” or “immunoreactive” portion of a BORIS isoform is meant any portion of the full-length BORIS isoform (e.g., SEQ ID NOs: 25-42) that can generate an immune response in a mammal in vivo, bind to an anti-BORIS antibody or autoantibody in vivo or in vitro, or comprises one or more epitopes of a BORIS isoform. As used herein, the term “portion” is synonymous with the term “fragment,” both of which are used to refer to contiguous part of a polypeptide comprising about 5 or more amino acids, such as about 10 or more, about 15 or more, about 20 or more, about 25 or more, or even about 30 or more amino acids). Of course, a full length BORIS isoform can provide the immunogenic portion; however, it can be more convenient to use a shorter portion of a BORIS isoform. One can determine whether any given portion of a BORIS isoform is immunogenic using routine techniques in view of the disclosures provided herein. For example, anti-BORIS antibodies to a BORIS isoform can be obtained from a mammal by introducing the BORIS isoform, or portion thereof, into the mammal and subsequently harvesting antibodies from the mammal using routine techniques. The given “test” portion of the BORIS isoform can be contacted with the anti-BORIS antibodies, and the binding affinity of the antibody to the portion of the BORIS isoform can be measured to determine whether the anti-BORIS antibodies bind to the given “test” portion of the BORIS isoform. If the antibodies bind to the test portion of the BORIS isoform, the test portion of the BORIS isoform is considered immunogenic. Other methods of determining whether a given portion of a BORIS isoform is immunogenic are available.
Suitable immunogenic portions of a BORIS isoform include the amino-terminal portion of a BORIS isoform (the “N-terminal domain”), defined as the region extending from the amino-terminal up to the zinc finger domain, or at least some portion thereof comprising about 100 or more amino acids (e.g., 200 or more, 250 or more, 300 or more, 400 or more, or 500 or more amino acids). Another suitable portion of a BORIS isoform polypeptide includes the carboxyl-terminal portion (the “C-terminal domain”), defined as the region starting after the zinc-finger domain and terminating at the carboxyl-terminus of BORIS, or at least some portion thereof comprising about 75 or more amino acids (e.g., about 100 or more, about 200 or more, about 300 or more, or about 400 or more amino acids).
The immunogenic portion of the BORIS isoform can be part of a larger polypeptide construct that comprises an amino acid sequence that is different from that of the native BORIS isoform. For instance, the immunogenic portion of a BORIS isoform can be part of a polypeptide construct comprising one or more different immunogenic portions of one or more different BORIS isoforms linked together, for example, by non-native amino acid sequences. Such a polypeptide construct might comprise, for instance, at least a portion of each of the N-terminal domain and the C-terminal domain of one or more different BORIS isoforms, as described herein. More preferably, the BORIS polypeptide construct comprises the entire N-terminal domain and C-terminal domain of one or more BORIS isoforms. It is further preferred that the BORIS polypeptide construct excludes any zinc finger domain of the BORIS isoform.
Even smaller portions of a BORIS isoform can provide an immunogenic portion, provided that a BORIS isoform epitope is present in the portion or fragment. By “epitope” is meant a sequence on an antigen that is recognized by an antibody or an antigen receptor. Epitopes also are referred to in the art as “antigenic determinants.” An immunogenic portion of the BORIS isoform can be less than about 660, 200, 150, 100, 60, 50, 30, 20, 15, or 12 amino acid residues in length, so long as it can be bound by an anti-BORIS antibody. The immunogenic portion of the BORIS isoform preferably comprises at least about 10, 11, or 12 amino acids; however, immunogenic portions of a BORIS isoform comprising fewer than 11 amino acids (e.g., about 4, 6, 8, or 10 or more amino acids) also are within the scope of the invention. Of course, the preferred number of amino acids also can be expressed in terms of ranges within any of the above-described preferred limits (e.g., 10-200 amino acids, 10-100 amino acids, 10-50 amino acids, 10-20 amino acids, etc.).
An immunogenic portion of a BORIS isoform also can be provided by a variant of the amino acid sequence of a BORIS isoform. As used herein, the term “variant” is used to refer to a sequence that is altered in the specific amino acid or nucleotide sequence, but retains the required function of the native sequence. With respect to the immunogenic portion of a BORIS isoform, a variant of the BORIS isoform retains the function of binding to an antibody to the BORIS isoform. BORIS isoform variants can be generated and characterized for their ability to bind with an anti-BORIS antibody or a functional fragment thereof (e.g., a Fab or F′(ab)2) using the information provided herein. For example, BORIS isoform variants can be generated using, for example, site-directed or random mutagenesis of a nucleic acid sequence encoding a BORIS isoform, as provided herein. The binding characteristics of the BORIS variant thus produced can be determined, for example, by measuring the binding affinity of antibodies to a BORIS isoform to the variant. Such antibodies can be obtained, for instance, from the serum of a mammal inoculated with a native BORIS isoform, or from the serum of a mammal with cancer.
A variant of a BORIS isoform desirably shares one or more regions of amino acid sequence identity with a native BORIS isoform. In this regard, the variant preferably comprises an amino acid sequence that is at least about 50% identical (e.g., at least about 60%, at least about 70%, at least about 80%, or at least about 90% identical) to the amino acid sequence of a native BORIS isoform. More preferably, the variant comprises an amino acid sequence that is at least about 75% identical (e.g., at least about 85%, or at least about 95% identical) to an amino acid sequence of a native BORIS isoform. Most preferably, the polypeptide comprises an amino acid sequence that is at least about 90% identical (e.g., at least about 95%, at least about 97%, or at least about 99% identical) to an amino acid sequence of a native BORIS isoform. As used herein, sequence identity is as determined using the well-known BLAST algorithms (e.g., BLASTp, BLAST 2.1, BL2SEQ, and later versions thereof)). Variants of a BORIS isoform capable of binding to anti-BORIS antibodies preferably have at least 5, 6, or 7 amino acid residues that are identical to the amino acid sequence of a native BORIS isoform over a window of eight amino acid residues.
Any immunogenic portion of a BORIS isoform can be used alone or in conjunction with other immunogenic portions of the same or different BORIS isoforms. The immunogenic portion of a BORIS isoform, whether used alone or in conjunction with other immunogenic portions of a BORIS isoform, also can be part of a larger polypeptide (e.g., inserted into (or otherwise attached to) another (i.e., “non-BORIS”) protein). Without intending to be bound by any particular theory, it is believed that different individuals will have immunogenic responses to BORIS isoforms based on the MHC molecules expressed on their antigen presenting cells (e.g., macrophages). Accordingly, the portion of BORIS isoforms that is immunogenic can vary from individual to individual. Moreover, an autoreactive antibody response directed against both the N-terminal and C-terminal domains of BORIS isoforms has been detected in some cancer patients. Thus, it is preferably the use of more than one immunogenic portion of the BORIS isoforms (e.g., more than one BORIS isoform epitope). When more than one immunogenic portion is used, the different immunogenic portions can be provided, for example, by several discontiguous polypeptides used simultaneously (e.g., two or more polypeptides each comprising a different immunogenic portion of a BORIS isoform) or by a single polypeptide comprising two or more different immunogenic portions of BORIS (e.g., linked by a non-native linker sequence).
In a preferred embodiment of the invention, two or more immunogenic portions of one or more BORIS isoforms are linked by a flexible linker amino acid sequence. Such a construct also can comprise an immunogenic portion of the BORIS polypeptide. Flexible linkers are used in the art to join two distinct polypeptides, such as, for example, in the construction of fusion or chimeric proteins. Thus, for example, an N-terminal domain portion and a C-terminal domain portion of one or more BORIS isoforms can be linked via a flexible linker sequence to form a single polypeptide molecule. The flexible linker can be any suitable amino acid sequence that can be used to join to separate polypeptide domains. In this regard, the flexible linker preferably comprises about 5 or more amino acids (e.g., about 6 or more, 7 or more, or 9 or more amino acids), more preferably about 10 or more amino acids (e.g., about 11 or more, 12 or more, or 14 or more amino acids), and most preferably about 15 or more amino acids (e.g., about 17 or more, 20 or more, or 25 or more amino acids). Linker sequences as well as methods for joining polypeptide domains using flexible linkers are known in the art (see, e.g., Imanishi et al., Biochem. Biophys. Res. Commun., 333(1), 167-73 (2005); Lin et al., Eur. Cytokine Netw., 15(3), 240-6 (2004)).
The BORIS isoforms or immunogenic portions thereof can be joined to other biomolecules, such as, for example, proteins, polypeptides, lipids, carbohydrates, prenyl, and acyl moieties, and nucleic acids. For instance, the BORIS isoforms or immunogenic portions thereof can be attached to a signaling moiety (also known as a detectable label). The identity and use of signaling moieties is well-known in the art. A signaling moiety is a molecule capable of indicating the presence of an analyte or reagent in a sample, usually after manipulation of the sample. Such manipulations often include incubating a sample and appropriate detection reagents under conditions allowing two moieties to bind together, if present, and then removing any of the labeled moiety from the sample via washing, filtration, or other suitable techniques. Other methods of working with signaling moieties are well-known in the art. Suitable signaling moieties include, but are not limited to, fluorescent molecules (e.g., green fluorescent protein), fluorescent quenchers, epitopes and haptens for antibodies that do not recognize BORIS (e.g., the well-known FLAG epitope), enzymes (e.g., chromogenic or luminescent (such as horse radish peroxidase or β-galactosidase)), a nucleic acid that can be amplified or specifically hybridized to a probe, biotin, avidin or streptavidin, lectins and colloids. Methods for linking proteins with detectable labels and solid supports are well-known in the art.
The antibody or autoantibody detected in accordance with a method of the invention preferably binds with greater affinity to BORIS or a BORIS isoform than to CCCTC-binding-factor (CTCF), or the antibody or autoantibody does not bind CTCF at all. Thus, the method of the invention preferably comprises detecting an anti-BORIS isoform antibody or autoantibody with a binding affinity for BORIS or a BORIS isoform that is greater than its binding affinity for CTCF. This is particularly advantageous where the portion of BORIS used comprises the zinc-finger domain of BORIS. More preferably, the dissociation constant (Kd) of binding under standard conditions between the antibody detected by the method of the invention and BORIS is at least 10-fold less, more preferably at least 100-fold less, or even 1000-fold less than the Kd of binding between the same antibody and CTCF. In some embodiments, binding of antibodies in a patient's serum to CTCF can be used as a negative control. When antibodies or autoantibodies to a particular BORIS isoform are desired, the method preferably comprises detecting such an antibody or autoantibody with a greater binding affinity for the desired target BORIS isoform than for other BORIS isoforms.
In a preferred embodiment, the method of detecting cancer or method of detecting anti-BORIS antibodies includes determining the class and/or subclass of the antibodies present in the patient's body, or sample derived therefrom, that are reactive with BORIS. One of ordinary skill in the art will appreciate that the five major human immunoglobulin classes (or “isotypes”) are immunoglobulin M (i.e., IgM), IgD, IgG, IgA, and IgE, which are typically defined by the structure of the constant regions of the antibody heavy chain. The light chain of a human antibody molecule is typically classified in the art as either a lambda (λ) chain or a kappa (κ) chain. IgG antibodies can be subdivided further into four subtypes (i.e., IgG1, IgG2, IgG3, and IgG4), whereas IgA antibodies typically are subdivided into two subtypes (i.e., IgA1 and IgA2). It is well-known in the art how to determine the class and subclass of isolated or purified antibodies. For example, BORIS-reactive antibodies can be isolated from a human's serum by immunochromatography. Wells of microtiter plates can be coated with 10 μg/ml of anti-human immunoglobin overnight at 4° C. After blocking with 5% BSA, the plates are reacted with 10 μg/ml of a monoclonal antibody or purified isotype controls, at ambient temperature for two hours. The wells can then be reacted with human IgG1-specific, IgG2-specific, IgG3-specific or IgG4-specific or human IgM-specific alkaline phosphatase-conjugated probes. After washing, the plates can be developed with a luminogenic or chromogenic substrate and analyzed for light or color development.
The methods of detecting a disease and detecting abnormal BORIS expression can be used in different ways. For example, the method can be used simply to establish the existence of a disease state for the purposes of diagnosis or screening. In addition, the method can be used, for example, to monitor the status (e.g., progression or regression) of a disease state, such as by comparing the level of anti-BORIS antibodies (or BORIS expression levels) from different samples over time. Such a use would be helpful in monitoring the response of patients to a particular therapeutic regimen.
In a related aspect, the invention also provides a method of detecting abnormal BORIS expression for purposes other than the detection of disease. The detection of abnormal BORIS expression can be used for any suitable purpose, such as for prognosticating, monitoring, or researching diseases characterized by abnormal gene expression, especially abnormal BORIS expression, including without limitation hyperproliferative diseases such as cancer. For instance, the methods of detecting abnormal BORIS expression is can be used to monitor the effect of drugs and other therapies on various diseases, especially various cancers described herein, in connection with the development of new or existing drugs or therapies, or as part of an established therapy regimen. Also, the methods of detecting abnormal BORIS expression can be used to generate BORIS expression profiles, which, in turn, can be used in accordance with methods of screening for, detecting, diagnosing, prognosticating, monitoring, or researching disease. The method of detecting abnormal BORIS expression can comprise testing for the expression of one or more BORIS isoforms comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42, or testing for the expression of one or more BORIS isoform mRNA transcripts comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-24, in the tissue of a mammal that does not express the BORIS isoform in the absence of a disease. All other aspects of the method of detecting abnormal BORIS expression are as described with respect to the method of detecting a disease associated with abnormal BORIS expression.
The mammal used in conjunction with the methods described herein can be any suitable mammal, such as dogs, cats, cows, goats, pigs, mice, rats, guinea pigs, rabbits, gerbils, monkeys, and hamsters. The mammal preferably is a human.
Isolated or Purified Polypeptide, Nucleic Acid, Antibody, Cell, and Composition
The invention provides an isolated or purified polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42, or an immunogenic portion thereof. The immunogenic portion of a BORIS isoform can comprise, consist essentially of, or consist of any of those immunogenic portions described herein as useful in conjunction with the method of detecting a disease or method of detecting an anti-BORIS autoantibody. The term “consisting essentially of” is used herein to mean that the polypeptide cannot comprise any other biologically active amino acid sequence, but can contain other non-biologically active sequences or other components such as regulatory or signal sequences, reporter constructs, linker molecules, targeting or delivery components, and the like.
If desired, the isolated or purified polypeptide can be modified, for instance, by glycosylation, amidation, carboxylation, or phosphorylation, or by the creation of acid addition salts, amides, esters, in particular C-terminal esters, and N-acyl derivatives of the polypeptide molecules of the invention. The polypeptide molecules also can be dimerized or polymerized. Moreover, the polypeptide molecules can be modified to create polypeptide derivatives by forming covalent or non-covalent complexes with other moieties in accordance with methods known in the art. Covalently-bound complexes can be prepared by linking the chemical moieties to functional groups on the side chains of amino acids comprising the polypeptides, or at the N- or C-terminus.
The isolated or purified polypeptide can be manufactured using any suitable method. In this regard, nucleic acid sequences encoding BORIS isoforms or immunogenic portions thereof can be synthetically produced using, for example, the nucleic acid sequences provided herein, and expressed in an appropriate host cell, thereby resulting in production of a BORIS isoform or immunogenic portion thereof. Alternatively, BORIS isoforms and immunogenic portions thereof can be synthesized using, for example, the amino acid sequences disclosed herein and protein synthesis methods known in the art. Alternatively, BORIS isoforms can be isolated from a mammal, and, as desired, immunogenic portions thereof can be generated using proteases that cleave within the full-length BORIS isoforms. As discussed above, BORIS isoforms or immunogenic portions thereof can be labeled with a signaling moiety or detectable label, or linked to a solid support.
The invention also provides an isolated or purified nucleic acid comprising, consisting essentially of or consisting of a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42, or an immunogenic portion thereof, as well as an isolated or purified nucleic acid comprising, consisting essentially of, or consisting of a nucleotide selected from the group consisting of SEQ ID NOs: 1-24. The term “consisting essentially of” is used herein to mean that the nucleic acid cannot comprise any other sequence that codes for a biologically active protein, but can contain other nucleic acid sequences or other components such as regulatory sequences, reporter constructs, linker molecules, and the like.
The present invention also provides a vector comprising an above-described isolated or purified nucleic acid molecule. A nucleic acid molecule as described above can be cloned into any suitable vector and can be used to transform or transfect any suitable host. The selection of vectors and methods to construct them are commonly known to persons of ordinary skill in the art and are described in general technical references (see, in general, “Recombinant DNA Part D,” Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)).
Suitable vectors include those designed for propagation and expansion or for expression or both. Examples of suitable vectors include plasmids, phagemids, cosmids, viruses, and other vehicles derived from viral or bacterial sources. Preferably, the vector is a viral vector and is selected from the group consisting of an adenovirus, adeno-associated virus, retroviruses, SV40-type viruses, polyoma viruses, Epstein Barr viruses, papillomaviruses, herpes virus, vaccinia virus and polio virus. Most preferably, the vector is an adenoviral vector.
When an adenoviral vector is used in the context of the present invention, the adenoviral vector can be derived from any serotype of adenovirus. Adenoviral stocks that can be employed as a source of adenovirus can be amplified from the adenoviral serotypes 1 through 51, which are currently available from the American Type Culture Collection (ATCC, Manassas, Va.), or from any other serotype of adenovirus available from any other source. For instance, an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, and 35), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and 41), or any other adenoviral serotype. Preferably, however, an adenovirus is of serotype 2, 5 or 9. However, non-group C adenoviruses can be used to prepare adenoviral vectors for delivery of one or more non-native nucleic acid sequences to a desired tissue. Preferred adenoviruses used in the construction of non-group C adenoviral vectors include Ad12 (group A), Ad7 (group B), Ad30 and Ad36 (group D), Ad4 (group E), and Ad41 (group F). Non-group C adenoviral vectors, methods of producing non-group C adenoviral vectors, and methods of using non-group C adenoviral vectors are disclosed in, for example, U.S. Pat. Nos. 5,801,030; 5,837,511; and 5,849,561 and International Patent Applications WO 97/12986 and WO 98/53087.
In preferred embodiments, the adenoviral vector of the present invention is deficient in one or more replication-essential gene functions. Regions contained within the adenoviral genome which are essential for replication include E1a, E1b, E2, E4, and L1-L5. By “deficient” is meant a disruption contained within at least one of the above-mentioned regions such that the gene product encoded by the region is produced in a reduced amount as compared to normal levels. Suitable disruptions include point mutations, substitutions, deletions, insertions, and inversions. Typically, the adenoviral vector is deficient in one or more replication-essential gene functions of the E1a, E1b, E3 and/or E4 region.
A nucleic acid sequence encoding a marker protein, such as green fluorescent protein or luciferase also can be present in the vector. Such marker proteins are useful in vector construction and determining vector migration. Marker proteins also can be used to determine points of injection in order to efficiently space injections of a vector composition to provide a widespread area of treatment, if desired. Alternatively, a nucleic acid sequence encoding a selection factor, which also is useful in vector construction protocols, can be part of the adenoviral vector.
Negative selection genes may be incorporated into any of the above-described vectors. A preferred embodiment is an HSV tk gene cassette (Zjilstra et al., Nature, 342: 435 (1989); Mansour et al., Nature, 336: 348 (1988); Johnson et al., Science, 245: 1234 (1989): Adair et al., PNAS, 86: 4574 (1989); Capecchi, M., Science, 244: 1288 (1989), incorporated herein by reference) operably linked to a viral promoter in a viral vector. The tk expression cassette (or other negative selection expression cassette) is inserted into the viral genome, for example, as a replacement for a substantial deletion of a non-essential viral gene. Other negative selection genes will be apparent to those of skill in the art.
The vector of the present invention can comprise a native or non-native regulatory sequence operably linked to an isolated or purified nucleic acid molecule as described above. If more than one nucleotide sequence is included in the nucleic acid molecule, each sequence can be operably linked to its own regulatory sequence. The “regulatory sequence” is typically a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the nucleic acid to which it is operably linked. The regulatory sequence can, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus, Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus and other retroviruses, and Herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. The promoters useful as regulatory sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art and can be used in the context of the invention, when desired. The selection of promoters, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the skill in the art. Similarly, the combining of a nucleic acid molecule as described above with a promoter is also within the skill in the art.
The term “operably linked” as used herein can be defined when a nucleic acid molecule and the regulatory sequence are covalently linked in such a way as to place the expression of the nucleotide coding sequence under the influence or control of the regulatory sequence. Thus, a regulatory sequence would be operably linked to a nucleic acid molecule if the regulatory sequence were capable of effecting transcription of that nucleic acid molecule such that the resulting transcript is translated into the desired protein or polypeptide.
The present invention further provides a cell (i.e., a host cell) comprising an isolated or purified nucleic acid molecule or a vector as described above, preferably a cell that expresses a BORIS isoform or immunogenic portion thereof, as described herein. Examples of host cells include, but are not limited to, a prokaryotic or eurkaryotic host cell. Prokaryotic cells include those derived from E. coli, B. subtilis, P. aerugenosa, S. cerevisiae, and N. crassa. Preferably, the host cell is derived from a mammal, such as a human.
An antibody (polyclonal or monoclonal) to a BORIS isoform or immunogenic portion thereof also is contemplated as part of the invention, as well as a cell line that produces a monoclonal antibody to a BORIS isoform or immunogenic portion. Such “hybridoma cell lines” desirably produce a monoclonal antibody that is specific for a BORIS isoform. Methods of making polyclonal antibodies and hybridomas are known in the art (see, e.g., Roitt I., Immunology, 4th Ed., Mosby, N.Y. (1996)). Typically, the antibody will be specific for a region of a BORIS isoform or a region of an immunogenic portion of a BORIS isoform. Typically, the region will be the N- or C-terminal portion of the BORIS isoform. Alternatively, the antibody can be specific for a zinc finger region of a BORIS isoform. Such an antibody will have a greater affinity for zinc finger regions of a BORIS isoform as compared to other proteins containing similar zinc finger regions (e.g., CTCF); thus being able to distinguish between the two molecules. Antibodies of the invention can be employed for both diagnostic and therapeutic applications as they are described herein.
The invention further provides a composition comprising an isolated or purified polypeptide, nucleic acid, or antibody as described herein and a carrier, preferably a pharmaceutically acceptable carrier. The phrase “pharmaceutically acceptable carrier,” as used herein, refers to a carrier that does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient. The pharmaceutical compositions of the present invention can be in a variety of forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, injectable and infusible solutions. Inhalable preparations, such as aerosols, are also included. Preferred formulations are those directed to oral, intranasal and parenteral applications, but it will be appreciated that the preferred farm will depend on the particular diagnostic or therapeutic application. The methods for the formulation and preparation of pharmaceutical compositions are well known in the art and are described in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985), The Merck Index, 11th ed., (Merck & Co. 1989), and Langer, Science, 249, 1527-1533 (1990).
The composition can comprise more than one active ingredient. Alternatively, or additionally, the composition can comprise another pharmaceutically active agent or drug. For example, when treating cancer, other anticancer compounds can be used in conjunction with the composition of the present invention and include, but are not limited to, all of the known anticancer compounds approved for marketing in the United States and those that will become approved in the future. See, for example, Table 1 and Table 2 of Boyd, Current Therapy in Oncology, Section 1. Introduction to Cancer Therapy (J. E. Niederhuber, ed.), Chapter 2, by B.C. Decker, Inc., Philadelphia, 1993, pp. 11-22. More particularly, these other anticancer compounds include doxorubicin, bleomycin, vincristine, vinblastine, VP-16, VW-26, cisplatin, carboplatin, procarbazine, and taxol for solid tumors in general; alkylating agents, such as BCNU, CCNU, methyl-CCNU and DTIC, for brain or kidney cancers; and antimetabolites, such as 5-FU and methotrexate, for colon cancer.
The compounds and compositions described herein can be used for any purpose. In addition to being useful in the method of detecting a disease and method of detecting abnormal BORIS expression, the compounds and compositions described herein can be used for other purposes, such as for inducing an immune response in a mammal. Such immune responses have multiple uses. For example, antibodies and other immunity-related molecules specific for BORIS can be isolated and used for research, control reagents useful in a method for detecting BORIS expression in a mammal, and as a method for destroying cancer cells that are present or could arise in a mammal. In this regard, the invention provides, as a related aspect, a method of inducing an immune response in a mammal comprising administering to a mammal a BORIS polypeptide as defined herein. Suitable methods of administration are known in the art. All other aspects of the method of inducing an immune response are as previously described herein.
Method of Treating a Disease Associated with Abnormal BORIS Expression
The invention provides a method of treating or preventing a disease associated with abnormal BORIS expression in a mammal comprising administering to a mammal that exhibits abnormal BORIS expression an inhibitor of a BORIS isoform. The inhibitor of the BORIS isoform can be any compound and/or molecule or any other agent capable of inhibiting the normal function of the BORIS isoform, or capable of inhibiting the expression of the BORIS isoform at the DNA or RNA level. Typically, the inhibitor of BORIS is a small molecule, an antibody, an antisense molecule, or a ribozyme molecule. It is also conceivable to provide an inhibitor of a BORIS isoform that comprises a molecule (e.g., a zinc finger binding protein) that recognizes zinc finger binding domains specific for a BORIS isoform and can therefore initiate its inhibition. It will be understood that when such zinc finger binding proteins are used, these molecules will be employed to specifically recognize zinc finger binding domains of a BORIS isoform as compared to other proteins comprising similar zinc finger binding domains (e.g., CTCF), such that the normal function of these similar proteins is not inhibited. Methods of identifying these inhibitors are well known in the art and can be accomplished without any undue experimentation using a variety of in vitro assays.
By “treating” is meant the amelioration of a pathologic state of any symptom thereof, in whole or in part. By “preventing” is meant the protection, in whole or in part, against a particular pathologic state, including, but not limited to, the prevention of the onset of any one or more symptoms of a pathologic state. One of ordinary skill in the art will appreciate that any degree of protection from, or amelioration of, a pathologic state is beneficial to a mammal.
According to a preferred aspect of the invention, the method of treating or preventing a disease associated with abnormal BORIS expression in a mammal comprises administering a short interfering RNA (siRNA) molecule to a mammal afflicted with a disease associated with abnormal BORIS expression, wherein the siRNA molecule comprises a sequence of at least about 10 contiguous nucleotides, preferably at least about 15 nucleotides, or even at least about 20 nucleotides (typically 21 nucleotides) that is complimentary to a portion or fragment of a BORIS isoform mRNA transcript (e.g., an mRNA transcript comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-24). Methods of designing siRNA molecules are known in the art. Typically, the siRNA will have a 3′ dinucleotide overhang (preferably UU residues). Accordingly, the target site generally will be chosen to be a site with an appropriate dinucleotide at the start position, such as an “AA” dinucleotide along with the appropriate number of 3′ nucleotides. The siRNA, of course, will have the complimentary sequence.
According to another preferred aspect of the invention, method of treating or preventing a disease associated with abnormal BORIS expression in a mammal comprises administering an anti-BORIS isoform antibody to a mammal afflicted with a disease associated with abnormal BORIS expression, wherein the anti-BORIS isoform antibody selectively binds to a BORIS isoform polypeptide (e.g., a BORIS isoform polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42). Suitable anti-BORIS isoform antibodies, including BORIS isoform-specific antibodies, can be generated given the information provided herein and routine techniques, as previously described.
Suitably, the inhibitor will be administered as part of a composition comprising a carrier. Suitable carriers and routes of administration are as described with respect to the other aspects of the invention.
Kit and Array Useful for Detecting BORIS Expression
The invention provides a kit useful for detecting BORIS expression comprising (a) a probe set comprising one or more probes that bind to (i) a BORIS isoform polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42, (ii) an auto-antibody to a BORIS isoform polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42, or (iii) a BORIS isoform mRNA transcript comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-24, and (b) a reagent that facilitates the detection of the probe.
The probe set can comprise one or more one or more antibodies, one or more polypeptides, or one or more nucleic acids (i.e., polynucleotides) depending upon whether the target to be detected in the sample is a BORIS isoform (in which can an antibody probe is useful), an auto-antibody to BORIS (in which case a polypeptide probe is useful), or a BORIS isoform mRNA transcript (in which case a nucleic acid probe is useful). Probes that specifically bind the respective target molecules can be designed using routine techniques. The probe specifically binds a target BORIS isoform polypeptide, auto-antibody, or mRNA transcript with preference over other molecules in the sample, such that the probe can be used to differentiate between the target molecule and the other molecules in the sample.
Polynucleotide and polypeptide probes can be generated by any suitable method known in the art (see, e.g., Sambrook et al., supra). For example, polynucleotide probes that specifically bind to BORIS isoform mRNA transcripts can be created using the nucleic acid sequences of BORIS isoform mRNA transcripts themselves (as disclosed herein) by routine techniques (e.g., PCR or synthesis). By way of further illustration, a polynucleotide probe that binds to the mRNA transcript of a particular BORIS isoform can be provided by a polynucleotide comprising a nucleic acid sequence that is complementary to the mRNA sequence or a fragment thereof, or sufficiently complementary to the sequence or fragment thereof that it will selectively bind to the sequence (e.g., bind to the target mRNA transcript with greater affinity than to other mRNA transcripts in the sample). The exact nature of the polynucleotide probe is not critical to the invention; any probe that will selectively bind the mRNA target can be used. Typically, the polynucleotide probes will comprise about 10 or more nucleic acids (e.g., about 20 or more, 50 or more, or 100 or more nucleic acids). Generally, the probe will contain fewer than 50 nucleotides. Thus, for example, the polynucleotide probe can comprise, consist essentially of, or consist of a fragment of any of SEQ ID NOs: 1-24 or complement thereof. In order to confer sufficient specificity, the nucleic acid probe will have a sequence identity to a compliment of the target sequence of about 90% or more, preferably about 95% or more (e.g., about 98% or more or about 99% or more) as determined, for example, using the well-known Basic Local Alignment Search Tool (BLAST) algorithm (available through the National Center for Biotechnology Information (NCBI), Bethesda, Md.). More preferably, the probe will comprise no more than one or two base-pair mismatches with the target sequence.
Similarly, polypeptide probes that bind to the BORIS isoform polypeptides described herein can be created using the amino acid sequences of the BORIS isoforms, disclosed herein, and routine techniques. For example, antibodies or antibody fragments to the BORIS isoforms can be generated in a mammal using routine techniques, which antibodies can be harvested to serve as probes for the BORIS isoforms. The exact nature of the polypeptide probe is not critical to the invention; any probe that will selectively bind to the BORIS isoform target can be used. Preferred polypeptide probes include antibodies and antibody fragments antibodies or antibody fragments (e.g., F(ab)2′ fragments, single chain antibody variable region fragment (ScFv) chains, and the like). Antibodies suitable for detecting the biomarkers can be prepared by routine methods, and are commercially available. See, for instance, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Publishers, Cold Spring Harbor, N.Y., 1988.
The reagent that facilitates detection of the probe can be any suitable reagent known in the art. For example, the reagent can be a molecule or compound that can be used to label the probe or the target molecules in the sample before or after contacting the sample with the probe. Specific examples of such reagents include, without limitation, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles).
The kit can further comprise one or more BORIS expression profiles (e.g., the expression profile of one or more BORIS isoforms) corresponding to one or more types of diseases or cancers. The BORIS expression profile for a given type of disease or cancer can be generated, for example, by testing for the expression of the BORIS isoforms and/or the BORIS polypeptide in a mammal or population of mammals known to be afflicted with the disease or cancer, or by testing for the expression of BORIS isoforms and/or the BORIS polypeptide in a cell line representative of a specific cell line. Preferably, the BORIS expression profile is generated from a mammal or, more preferably, a population of mammals known to be afflicted with a particular disease. The BORIS expression profile can serve as a reference by which to compare the BORIS expression of a given mammal of an unknown disease state in order to determine whether the mammal, in fact, has a disease or propensity to develop a disease.
Preferably, the BORIS expression profiles are provided in the form of a database comprising the BORIS expression profile of one or more different types of cancer or other diseases associated with abnormal BORIS expression, wherein the database facilitates the comparison of a BORIS expression profile of a patient with the BORIS expression profile of one or more different types of cancer. Such databases that facilitate the comparison of the BORIS expression profile of a patient with the BORIS expression profile of one or more different types of cancer or other diseases include, for example, searchable databases, especially searchable electronic databases.
The probes of the probe set can be immobilized on a suitable substrate, so as to provide an array. In this respect, the invention also provides an array useful for the detection of BORIS expression in a mammal, the array comprising one or more probes immobilized on a substrate, wherein the probes bind to (i) a BORIS isoform comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42, (ii) an auto-antibody to a BORIS isoform comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-42, or (iii) a BORIS isoform mRNA transcript comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-24.
The substrate can be any rigid or semi-rigid support to which polynucleotides or polypeptides can be covalently or non-covalently attached. Suitable substrates include membranes, filters, chips, slides, wafers, fibers, beads, gels, capillaries, plates, polymers, microparticles, and the like. Materials that are suitable for substrates include, for example, nylon, glass, ceramic, plastic, silica, aluminosilicates, borosilicates, metal oxides such as alumina and nickel oxide, various clays, nitrocellulose, and the like.
The polynucleotide or polypeptide probes can be attached to the substrate in a pre-determined 1- or 2-dimensional arrangement, such that the pattern of hybridization or binding to a probe is easily correlated with the expression of a particular BORIS isoform. Because the probes are located at specified locations on the substrate, the hybridization or binding patterns and intensities create a unique expression profile, which can be interpreted in terms of the expression of particular BORIS isoforms.
The array can comprise other elements common to polynucleotide and polypeptide arrays. For instance, the array also can include one or more elements that serve as a control, standard, or reference molecule, such as a housekeeping gene or portion thereof (e.g., PBGD, GAPDH), to assist in the normalization of expression levels or the determination of nucleic acid quality and binding characteristics, reagent quality and effectiveness, hybridization success, analysis thresholds and success, etc. These other common aspects of the arrays or the addressable elements, as well as methods for constructing and using arrays, including generating, labeling, and attaching suitable probes to the substrate, consistent with the invention are well-known in the art. Other aspects of the array are as previously described herein with respect to the methods of the invention.
The kit or array can comprise a single probe for a single BORIS isoform polypeptide, mRNA transcript, or autoantibody. However, the kit or array advantageously comprises more than one probe, such as two or more, three or more, four or more, five or more, 10 or more, 15 or more, or even 20 or more different probes that each bind to a different BORIS isoform polypeptide, mRNA transcript, or autoantibody (or even a different probe for each of the BORIS isoform polypeptides, mRNA transcripts, or autoantibodies identified herein).
The kit or array can further comprise probes for polypeptides, mRNA transcripts, or autoantibodies other than BORIS isoform polypeptides, mRNA transcripts or autoantibodies. For example, it is desirable for the kit or array to comprise a probe for a BORIS polypeptide comprising the amino acid sequence of SEQ ID NO: 43, the mRNA transcript encoding such a BORIS polypeptide (e.g., an mRNA transcript comprising the nucleic acid sequence of SEQ ID NO: 44), or an autoantibody to such a BORIS polypeptide. Other probes also can be included, such as probes that bind to other tumor antigens or other genetic cancer markers. Nevertheless, it may be convenient in some instances to limit the total number of different probes for ease of use. Thus, for instance, the kit or array can comprise less than about 1000 different probes, or less than about 500 different probes, such as less than about 100 different probes, or even less than about 50 different probes (e.g., less than about 30 different probes).
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
EXAMPLE 1
This example illustrates the identification of multiple BORIS isoforms in the human testes.
RT-PCR was used to identify and sequence the mRNA transcripts of 24 different mRNA splice variants of the BORIS gene expressed in the human testes. The 24 mRNA splice variants are depicted in Figures A, B, C, and D, which encode 18 different BORIS isoform polypeptides. The nucleotide sequences of the mRNA splice variants, as well as the amino acid sequences encoded by the mRNA splice variants, are set forth in Table 1.
EXAMPLE 2
This example illustrates the generation of isoform-specific anti-BORIS antibodies, and the use of such antibodies to identify BORIS isoforms in the human testes.
Isoform-specific anti-BORIS antibodies were generated to three different BORIS isoforms (SEQ ID NOs: 25, 27, and 32) by immunizing rabbits with synthetic peptides specific to each isoform. In particular, peptides CKYASVEVKPFLDLKLHGILVEAAVQVTPSVTNSRI (SEQ ID NO: 45) and CYKQAFYYSYKIYIGNNMHSLL (SEQ ID NO: 46) were used to develop antibodies to the isoform comprising SEQ ID NO: 25; peptides CLLGSSDSHASVSGAGITDARHHA (SEQ ID NO: 47) and CITDARHHAWLIVLLELVEMGFYHVSHS (SEQ ID NO: 48) were used to generate antibodies to the isoform comprising SEQ ID NO: 27; and peptides CPPGLHHPKAGLGPEDPLPGQLRHTAG (SEQ ID NO: 49) and GQLRHTTAGTGLSSLLQGPLC (SEQ ID NO: 50) were used to generate antibodies to the isoform comprising SEQ ID NO: 32. The rabbits were immunized with the peptides conjugated to keyhole limpet hemocyanin (KLH). Thereafter, peptide-specific antibodies were affinity purified on a column using the same peptides. The isoform-specific anti-BORIS antibodies were successfully used to detect the three different BORIS isoforms in the tissue of the human testes.
EXAMPLE 3
This example demonstrates the expression pattern of BORIS isoforms in the NCI-60 cell lines.
RT-PCR was used to test for the expression of six different BORIS isoforms in each of the NCI-60 cell lines. The NCI-60 cell line panel is considered to be representative of the vast majority of human cancer types. The results are presented in Table 1, wherein a “+” indicates positive expression, and a blank indicates no expression.
As illustrated by the results in Table 2, each of the NCI-60 cell lines showed expression of at least one isoform of BORIS. Moreover, different cancers exhibited different BORIS isoform expression patterns. These results show that BORIS isoform expression patterns can be used to differentiate between different types of cancers.











TABLE 1







mRNA Splice
Nucleotide
Amino Acid



Variant
Sequence
Sequence









BORIS A1
SEQ ID NO: 1
SEQ ID NO: 43



BORIS A2
SEQ ID NO: 2
SEQ ID NO: 43



BORIS C1
SEQ ID NO: 3
SEQ ID NO: 43



BORIS A4
SEQ ID NO: 4
SEQ ID NO: 25



BORIS C2
SEQ ID NO: 5
SEQ ID NO: 25



BORIS B1
SEQ ID NO: 6
SEQ ID NO: 26



BORIS C3
SEQ ID NO: 7
SEQ ID NO: 27



BORIS B2
SEQ ID NO: 8
SEQ ID NO: 28



BORIS B3
SEQ ID NO: 9
SEQ ID NO: 29



BORIS C4
SEQ ID NO: 10
SEQ ID NO: 30



BORIS C5
SEQ ID NO: 11
SEQ ID NO: 31



BORIS A5
SEQ ID NO: 12
SEQ ID NO: 32



BORIS A6
SEQ ID NO: 13
SEQ ID NO: 33



BORIS B4
SEQ ID NO: 14
SEQ ID NO: 34



BORIS B5
SEQ ID NO: 15
SEQ ID NO: 35



BORIS C6
SEQ ID NO: 16
SEQ ID NO: 36



BORIS B6
SEQ ID NO: 17
SEQ ID NO: 37



BORIS B7
SEQ ID NO: 18
SEQ ID NO: 37



BORIS C7
SEQ ID NO: 19
SEQ ID NO: 38



BORIS C8
SEQ ID NO: 20
SEQ ID NO: 39



BORIS C9
SEQ ID NO: 21
SEQ ID NO: 38



BORIS F6
SEQ ID NO: 22
SEQ ID NO: 40



BORIS F7
SEQ ID NO: 23
SEQ ID NO: 41



BORIS A3
SEQ ID NO: 24
SEQ ID NO: 42



BORIS
SEQ ID NO: 44
SEQ ID NO: 43





























TABLE 2








K562
MOLT-4
CEM
RPMI8226
HL-60
SF-295
SF-539
SNB-19
HS578T
MDAMB435
COLO-205
HCC-2998





BORISA4/C2
+
+
+

+
+

+
+
+

+


BORIS C3
+


BORIS A5
+





+


+
+


BORIS B1
+


BORIS F6
+


+


BORIS F7
+


+






















HCT-116
HCT-15
HT-29
KM12
SW-620
A549
EKVX
HOP-62
HOP-92
NCI-H322
NCI-H226





BORISA4/C2
+

+
+
+
+
+
+
+
+
+


BORIS C3






+
+


BORIS A5
+



+

+
+


BORIS B1



+
+

+
+


BORIS F6




+


BORIS F7









+






















NCI-H23
NCI-H460
NCI-H522
LOX
M14
MALME3M
SK-MEL-2
SK-MEL28
SK-MEL-5
UACC-257
UACC-62





BORISA4/C2
+
+
+
+

+
+
+
+
+
+


BORIS C3





+




+


BORIS A5




+
+


+
+
+


BORIS B1


BORIS F6


BORIS F7










+

























ARD-RES
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
786-0
ACHN
RXF-393
SN12C
TK-10
UO-31
PC-3







BORISA4/C2
+

+
+
+
+
+
+
+
+
+



BORIS C3
+



+



BORIS A5
+



+



BORIS B1
+



+



BORIS F6
+



+



BORIS F7
+
+


+
+





+










All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Wherever the invention is described with reference to open-ended terms (e.g., comprising), it is specifically contemplated that a qualified or closed-ended term (e.g., consisting essentially of or consisting of) can be used instead. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

## Claims

1. An isolated or purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-42.

2. A method of detecting a cancer characterized by abnormal BORIS expression in a mammal suspected of having cancer, wherein the expression of the one or more polypeptides in the tissue or body fluid is upregulated as compared to expression of the one or more polypeptides, which method comprises testing for the expression of one or more of the polypeptides of claim 1 in a tissue or body fluid of a mammal in the absence of cancer.

3. The method of claim 2, wherein testing for the expression of the one or more polypeptides comprises contacting a sample of body fluid or tissue of the mammal with an antibody to the one or more polypeptides, and detecting the binding of the antibody with one or more polypeptides from the sample.

4. The method of claim 2, wherein testing for the expression of the one or more polypeptides comprises detecting the presence of one or more auto-antibodies to the one or more polypeptides.

5. The method of claim 4, wherein detecting the presence of one or more auto-antibodies to the one or more polypeptides comprises contacting a sample of body fluid or tissue of the mammal with a probe comprising an immunogenic portion of the amino acid sequence of the one or more polypeptides, and detecting the binding of the probe with an auto-antibody from the sample.

6. The method of claim 2, wherein the method comprises testing for the expression of two or more different polypeptides, each of which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-42.

7. The method of claim 2, wherein the method further comprises comparing the expression of the one or more polypeptides in the mammal with a control.

8. The method of claim 7, wherein the control is a BORIS expression pattern of a mammal known to be afflicted with a particular cancer associated with abnormal BORIS expression.

9. A kit for the detection of BORIS expression in a mammal, which kit comprises
(a) a probe set comprising one or more probes that bind to (i) a BORIS isoform polypeptide, or (ii) an auto-antibody to a BORIS isoform polypeptide, wherein the BORIS isoform polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-42, and
(b) a reagent that facilitates the detection of the probe.

10. The kit of claim 9, wherein the probe set comprises one or more auto-antibodies to a BORIS isoform polypeptide.

11. The kit of claim 9, wherein the probe set comprises one or more BORIS isoform polypeptides.

12. The kit of claim 9, wherein the kit further comprises a BORIS expression profile of one or more types of cancer.

13. An antibody or antibody fragment that binds to the polypeptide of claim 1.

14. A composition comprising the antibody or antibody fragment of claim 13 and a carrier.

15. A method of treating or preventing a cancer associated with abnormal BORIS expression in a mammal comprising administering the antibody or antibody fragment of claim 13 to a mammal afflicted with a cancer associated with abnormal BORIS expression, wherein the antibody selectively binds to a BORIS isoform polypeptide.

16. A cell comprising the polypeptide of claim 1.

17. The method of claim 2, wherein the cancer is testes cancer.